This invention relates to mass spectrometry analysis, and methods and devices for increasing throughput and enhancing the quality of results obtained thereby and particularly relates to the precursor ionization, especially protonation, of subject compounds for analysis.
In basic mass spectrometry, a molecule is bombarded with an electron beam with sufficient energy to fragment it. The positive fragments which are produced (cations and radical cations) are accelerated in a vacuum through a magnetic field and are sorted on the basis of mass-to-charge ratio. Since the bulk of the ions produced in the mass spectrometer carry a unit positive charge, the value m/e is equivalent to the molecular weight of the fragment. The analysis of mass spectroscopy information involves the re-assembling of fragments, working backwards to generate the original molecule. Since the very process of ionizing the molecule causes the molecule to fragment, it has not been possible to directly generate the original molecule without fragment assembly.
In operation, a very low concentration of sample molecules is allowed to leak into the high vacuum ionization chamber where they are bombarded by a high-energy electron beam. The molecules fragment and the positive ions produced are accelerated through a charged array into an analyzing tube. The path of the charged molecules is bent by an applied magnetic field. Ions having low mass (low momentum) are deflected and collide with the walls of the analyzer and high momentum ions are not deflected enough and also collide with the analyzer wall. Ions having the proper mass-to-charge ratio, however, follow the path of the analyzer, exit through the slit and collide with the Collector to generate an electric current, which is amplified and detected. By varying the strength of the magnetic field, the mass-to-charge ratio which is analyzed can be continuously varied.
The output of the mass spectrometer shows a plot of relative intensity vs the mass-to-charge ratio (m/e), with the most intense peak in the spectrum being designated the base peak and all others are relative thereto in intensity. The peaks themselves are usually represented as vertical lines.
Fragmentation is predictable and the ions which are formed reflect the most stable cations and radical cations that the molecule can form. The highest molecular weight peak observed in a spectrum typically represents the parent molecule, minus an electron, and is referred to as the molecular ion (M+). Fragments can be identified by their mass-to-charge ratio, or, more preferably by the mass which has been lost.
A common mass spectroscopy method utilized with identifying compounds and molecule characteristics, especially in drug screening applications, is HPLC (High Performance Liquid Chromatography) with the mass spectrometer being utilized as a detector after a choromatographic separation. In such method, samples are injected onto a reversed-phase HPLC column and eluted with a solvent into the source of an electrospray ionization/ion trap mass spectrometer. The source converts the liquid effluent into an aerosol and ionizes the solutes in the aerosol. Desolvated ions are drawn into the analyzer of the mass spectrometer and are collected in a trap. Once the trap is filled, its voltages are varied so ions leave in an orderly, mass-dependent manner and strike a detector. By calibrating the process with ions of known mass, the unknown masses of samples can be measured (LC/MS).
In addition, specific ions can be isolated from other ions in the trap and fragmented by collision-induced dissociation, and the masses of the fragments can be measured (LC/MS/MS). For example, for a peptide, most fragmentations will occur at peptide bonds, and so the fragmentation patterns contain information about the peptide""s sequence, which can be used to identify proteins.
While LC/MS is a valuable analysis tool there are certain deficiencies. Thus, for example, LC/MS does not always produce the parent ion, and this may accordingly necessitate an undue effort to assign a molecular structure. In addition, standards are required to give a percent composition (molar). Also chromatographic separation is needed before mass analysis. Finally, there is difficulty in providing a wide encompassing range of ionization, from small solvent molecules to large proteins.
It is highly desirable, but often difficult, to provide for quick screen pilot reactions, not only to determine if the desired product is present, but also to determine the amount produced. In areas where high throughput screening is being used, speeding up the assays can be effected by removing the need to perform separations which are a mainstay of LC/MS. Additionally, for analyzing drug substances and drug products, impurities should be much easier to identify and further quantitation of impurities should be performed without the need for standards.
To be useful, any new method of ionization has to overcome the two following seemingly fundamental limitations:
(1) The ability of a molecule to pick up a charge either in solution or from a plasma is dependent on the proton affinity (PA) of the neutral molecule compared to the proton affinity of the solvent or gas. In other words, if a molecule does not have a high PA, it will not pick up a charge and will not be detected. This leads to different levels of response for different molecules and inconsistencies in measurements.
(2) The amount of energy deposited during the ionization and from collisions with neutral gas molecules raises the chance of fragmentation with concomitant increase in difficulty in reconstructing the molecule and determination of the parent molecule.
The primary cause of these limitations is that ions being created exist temporarily in a high pressure environment. As a result, there are collisions that lead to heating and the opportunity for charge exchange or ion molecule reactions. While it may be considered possible to obviate this problem by performing the ionization at low pressure, to limit the number of collisions with other gas molecules, the addition or removal of a proton (Chemical Ionization) or the removal of an electron (Electron Impact Ionization) is sufficiently exothermic, in itself, so as to cause covalent bonds to dissociate and in a high pressure ionization source, collisions with neutral gasses allow the energy to be removed from the ions before they can fragment. There is thus no ideal pressure for ionization without charge exchange or ion molecule reactions.
It is therefore an object of the present invention to provide a method and device for molecule ionization for analysis of the molecule such as with mass spectroscopy but with minimized or without charge exchange or ion molecule reaction.
It is yet another object of the present invention to provide a means to enhance consistency between measurements of different molecules with mass spectrometers.
It is still yet another object of the present invention to provide such means whereby molecules do not fragment with ionization or collision.
Generally the present invention comprises a method and device for effecting said method, for analyzing full molecules by mass spectroscopy without fragmentation of the molecules normally resulting from ionization or collisions. In accordance with the present invention, molecules are ionized in a sufficiently cold environment wherein fragmentation collisions are minimized and wherein heat transfer means such as a heat bath remove heat generated by the ionization prior to fragmentation of the molecule thereby.
In a very highly preferred embodiment of the present invention droplets of liquid helium, cooled to just above absolute zero, are used as an environment for ionization. A liquid helium droplet provides the molecule with a collision-free environment and at the same time provides a highly efficient method for removing the internal energy generated during ionization.
A device (Helium Droplet Mass Spectrometerxe2x80x94HDMS), used in accordance with the method of the present invention comprises the elements of:
a) helium droplet or cluster source for production of near absolute zero temperature droplets of helium;
b) proton source for supply of ions to protonate a molecule to permit mass spectrometer analysis;
c) atmospheric pressure (AP) source for protonation or ionization of the molecule;
d) means for desolvation or removal of excess liquid helium; and
e) analysis means such as a mass spectrometer with selection means and detection means for analysis and detection of the full protonated molecules, or selection means and detection means for measurement of the accurate mass, or selection means and detection means for providing controlled fragmentation of the full protonated molecule for structural analysis.
These and other objects, features and advantages of the present invention will become more evident from the following discussion and drawings in which: